Abstract

This paper introduces the advances in both basic and applied
researches in tree genetic engineering in China. Some recommendations are made
for prioritizing future studies in tree genetic engineering in order to meet the
requirements of environmental improvement and the timber industries.

1. Advances in tree genetic engineering in China

Researches on transgenic forest trees started in China at the
end of 1980s. Since then, Chinese researchers have developed techniques of
tissue culture and genetic transformation for many trees species, such as
poplar, birch, eucalypt, larch, walnut, apple, citrus, Chinese goosebeery, etc.
They successfully applied tissue culture and genetic transformation in many tree
species [1,2,4,10,11], and successfully transferred Bt gene,
salty tolerance gene, drought tolerance gene, sterile gene, anti-ACC synthetase
and oxidase gene, etc, into poplar genome by using leaf-disc, ballistic and
other methods with regenerated transgenic plants or varieties
obtained.

1.1 Genetic engineering targeted at
insect resistance

The application of genetic engineering technology for insect
resistance breeding is considered a new approach of great interest to protecting
forest trees from insect damages. Some successes have been achieved.

Transgenic Populus nigra with Bt gene
resistant to leaf insect was commercialized. The research on transferring
Bt gene into Populus nigra started in Chinain 1989, and
transgenic plants were put into field trails in 1994. According to 4-year
results of the field trails, the transgenic Populus nigra plants were
significantly resistant to target insects. Insect population in both trees and
soil were decreased, and both transgenic and non-transgenic plants in the same
planting site were protected. In 1998, transgenic Populus nigra plants
were approved for environmental release at Manasi in Xinjiang by the
Bio-Engineering Security Committee of the Ministry of Agriculture, and approved
for environmental release in Beijing, Jilin, Shandong, Jiangsu, Henan, and
Shanxi provinces in 1999. Because of their rapid growth and high resistance to
the target insect, commercialization of the transgenic Populus nigra
plants were approved by the Gene Security Committee of the State Forestry
Administration in 2002. Up to now, one million of the transgenic Populus
nigra plants have been propagated and the plantations of transgenic plants
have been established.

Transgenic hybrid poplar 741 with bi-resistant insect genes
was approved for environmental release. In 1996, researchers transferred the
bi-vector resistant insect that contained the improved Bt Cry1Ac gene and
API gene to hybrid poplar 741 (Populus alba L.¡Á(P.
davidiana Dode + P. simonii Carr.)¡ÁP. tomentosa
Carr.), and identified that novel genes had transferred into poplar plants with
normal growth and development of transgenic plants[3]. Through a
5-year experiment, the Forest Bio-Engineering Security Committee approved the
environmental release of 6 clones of transgenic hybrid poplar 741 in 2001. So
far, 400,000 plants of transgenic poplar 741 have been propagated and a breeding
system and a trail plantation have been established to provide new elite
varieties.

Transgenic poplar plants resistant to trunk-insect
(longhorn beetle) Researchers have transferred the LcI and Bt
genes into poplar. The imago of Anoplophora glabripennis
(Motschulsky) was fed with the leaves of transgenic plants. More than 30 clones
were obtained. Propagation of some transgenic plants and field trails has been
conducted [17].

Chinese researchers not only successfully transferred the
Bt gene into poplar, larch and walnut, but also transferred CpTI, OC-I,
and AaIT genes into some varieties of poplar [7,8]. The
preliminary study on transferring the insect resistant gene into Betula
platyphylla was also started [12].

1.2 Transgenic experiments for drought
and salty resistance

Up to now, few researches on genetic engineering targeted at
drought and salty tolerance, mainly because the plant response to drought and
salty is a multi-component-system, and the physiological mechanism controlled by
multi-genes has not been known clearly.Chinese researchers transferred
SacB and HVA1 genes into poplar through agrobacterium
tumefaciens-mediated method, and obtained regenerated transgenic plants.
Zhan et al transferred BetA into Populus simonii × P.
nigra by agrobacterium tumefaciens mediated method using sterile
seedling leaf as explants, and the results of Southern-blot analysis indicated
that the novel gene was integrated into target genome [14]. In
addition, the experiments of transferring BADH and GUTD genes into poplar and
transferring Ip3 and Ip5 genes into larch are in
progress.

1.3 Transgenic engineering for disease
resistance

Chinese researchers have made some meaningful attempts,
although many pathogenies are not isolated and identified, causing difficulties
in transgenic engineering for disease resistance. Researchers transferred the
rabbit alexin gene NP-1 into Populus tomentosa Carr. by agrobacterium
tumefaciens-mediated method, and identified by Southern analysis that the
novel gene had integrated into transgenic plant genome, and results from the
experiment of inhibitory in vitro indicated that the transgenic plants could
restrict the growth of many kinds of microbe, such as Bacillus
subtilis, agrobacterium tumefaciens, and Rhizoctonia solani[13]. Researchers also successfully transferred some novel genes into
papaya by agrobacterium tumefaciens mediated
[15,16,19,20].

1.4 Transgenic experiments for quality
improvement

For preventing senescence of forest trees, researchers used
the anti-RNA technology to control the ethylene biosyntheses. They transferred
the anti-fragment of ACC oxidase gene into Populus deltiodes, and the
results of Southern analysis showed that a single copy of novel gene was
integrated into transgenic plant genome, the ethylene bio-synthesis of
transgenic plants was depressed, and the ethylene produced by their seedlings
was 28% less than by the control plants [18]. The genetic
transformation studies for decreasing the content of lignin in poplar and larch
are being conducted, and a few transgenic clones were obtained.

1.5 Sterile engineering

Researchers attempted to study the novel gene escape from
transgenic trees, they transferred TA29-BARNASE gene into Populus
deltiodes, and identified by PCR, Southern blot and Nouthern blot analysis
that the novel gene was integrated into transgenic plant genome
[6].

1.6 Field trails of transgenic
plants

In abroad, except that the virus resistance transgenic papaya
was commercialized, almost all other transgenic trees were under small scale of
field trails. In China, the environmental release of transgenic Populus
nigra with Bt gene is being conducted at 8 sites in Beijing, Jilin,
Henan, Shandong, Xinjiang, Shanxi, and Jiangsu provinces with a total area of 80
hm2.

1.7 Transgenic safety
evaluation

The research on transgenic safety evaluation started in China.
Chinese researchers have studied the effects of Bt genetransgenic
Populus nigra on un-targeted insects, microbes in soil, mammals and human
beings, and obtained some results. More researches on environmental release of
Bt gene related to environmental security are under way. Researches in
this field are considered useful for proper application of transgenic forest
trees, and security evaluation system and related standards.

1.8 Basic research in gene
cloning

Chinese researchers have made great efforts in gene cloning in
order to win intellectual property right of our own. Researchers cloned the full
length of genome, ABREBP-type transcript factor genes from Caragana
korshinskii growing in desert with drought tolerance, and the cDNA full
length is being cloned. Liu et al. cloned the mtl-D gene related to salty
tolerance from E. coli, inserted it to binary vector and transferred into
a poplar cultivar Balizhuangyang by agrobacterium tumefaciens, the
transgenic plants grew very well in media containing 0.6% NaCl; PCR and Southern
blot analysis indicated that the novel gene was integrated into the target
genome and expressed [5]. Wei et al. cloned a COMT cDNA fragment,
encoding for methylase that relates to lignin biosynthesis pathway, from Chinese
White Poplar (Populus tomentosa) [9]. Researchers are
currently cloning CCoAOMT, PAL and CCR genes. They have identified a new species
of Bacillus thuringiensis, virulent to Ceram bycidae from the
natural cadaver of Ceram bycidae and from the resources of Bacillus
thuringiensis; and feeding experiment showed this new Bt could kill
50% imago of Ceram bycidae. The related fragment of this Bt gene
was then amplified using the special annex primer, and identified that the
fragment was really a kind of Bt gene resistant to Ceram bycidae.
According to the results of sequencing, the full length of this gene cloned and
the expression vector for transformation is being constructed.

2 Key areas for future tree genetic
engineering in China

Although many fast-growing tree species have been developed by
breeding and introduction in recent 20 years, fast growth has been over
emphasized while improvement of wood quality has been overlooked, resulting in
high cost of plantation management and largely constraining wood processing and
utilization. Overlooking of the resistance to biological or non - biological
stresses of trees in ecologically fragile environment in western China, had led
to very low survive rate of tree plantings and frequent disasters of
Cerambycidae, Apocheima cinerarius (Erschoff), Lymantria
dispar L., etc., large area of timber plantations and ecological protective
forests are losing their functions, all these indicate clearly that studies on
gene transformation of trees must be immediate action with no delay.

Considering the unique climate and nature of China, and new
threats from diseases and insects related to global warming, we suggest that the
following key fields be included in research programs in the near
future.

Among the transgenic studies, research on insect resistance
has been best conducted and achieved the best progress. However, all current
studies are focused on lepidopterous insects, and inadequate attention has been
paid to Cerambycidae, a trunk-insect that damages many tree species,
prevails widely and causes the most severe economic lost.

Cerambycidae has a wide range of host trees, including
poplar, willow, elm, maple, Sophora japonica, mulberry, etc. Damages to
poplar and pine are the most serious. As is well known, Bursaphelenchus
nematode has caused fatal damage to pine plantations, with an area of 74000
hm2, and its carrier was Monochamus alternatus Hope. The Three
North Shelterbelts Project, which attracts worldwide attention, covers 500
counties, but now Cerambycidae disaster (mainly caused by Anoplophora
glabripennis M.) has happened in more than 300 counties, covering an area of
268,000 hm2. More than 17 million cube meters, timber was destroyed,
causing a direct economic loss of up to 5 billion RMB. This disaster will spread
to natural forests if no effective measure was taken.

Trunk hosting makes Cerambycidae more difficult to
control. An important target for tree genetic engineering is to find out and
obtain the gene that could kill Cerambycidae effectively. Bacillus
thuringiensis is recognized as a kind of pathogeny microbes that are
harmless to human or livestock, non-polluting to environment, and protective
towards natural enemies. A feasible approach is to screen out and identify the
genes that could kill Cerambycidae from Bt, analyze the function
and structure of Bt gene and improve it, and conduct the transgenic
researches to obtain the tree species resistant to Ceram bycidae so that
the goal of controlling Cerambycidae could be reached.

2.2 Disease (rust disease and canker
disease) resistance

According to incomplete statistic, the occurrence of forest
insect and disease in China covered an area of 1 million hm2 in
1950s, 1.4 million hm2 in the 1960s, and up to 11 million
hm2 in the 1990s, the annual increase was 25%. The economic loss per
year reached more than 5 billion RMB, the disaster affected area was 8.2% of the
total forest area and 23.7% of the total plantation area. Disease and insects
have already been an important factor restricting the sustainable forestry
development in China. Among the diseases, canker and rust are the most serious
diseases. Disease decreases the quality of wood from timber plantations and
reduces productivity of economic forest, leading to serious damages and economic
loss.

The fungi-resistance genes mainly are those genes from the
inter- or outer- plant pathogenies, such as antagonistic protein gene and
virulent protein gene, the genes encoded motivating factors of pathogeny,
protease inhibitor genes of pathogeny and genes that controlling sensitive
response of host and pathogeny, etc. But at present, the effective genes
resistant to Botryosphaeria ribis and Melampsora
larici-populina Kleb have not been cloned. Therefore, we should first
conduct the gene clone experiments in genetic engineering program. It is
suggested that our government increase investment in this research
fields.

2.3 Stress (drought, salty, and
coldness) resistance

The stresses, such as salty, drought, waterlogging, unsuitable
temperature, strong sunlight, residue of pesticide, etc, restrict the range of
forest plantations, timber productivity and wood quality. For making full use of
the existing land, expanding forest plantations and improving timber
productivity and wood quality, breeding for stresses resistance is attracting
more and more attentions. In traditional breeding project, for lacking of the
resources of resistance and the knowledge of resistance mechanism, little
progress has been made in this field. However, the recent advance of
biotechnology has provided a new opportunity.

Because the mechanisms of drought and salty resistance in
plants are very complex and involve a series of variation of phenotype and
metabolism, the single gene transformation could only obtain partial resistance
in most cases. If the transgenic trees are expected to be able to grow in
drought area or near the beach, and to be irrigated with sea water, the
followings are needed: first, transferring multi-genes; second, using the
advanced methods, such as chloroplast transformation, and enhancing novel gene
expression; third, transferring with transcript factors. Transcript regulation
factors, through the mediation of related cis-action elements, could induce many
gene expressions and enhance plant resistance to drought, salty and cold.
Transgenic trees with drought and salty resistance genes will have a broad
application in China, but research in this field is still weak and needs further
strengthening.

2.4 Wood quality
improvement

At present, the genetic engineering of lignin biosynthesis
appears to be a promising forest biotechnological application to improve wood
quality. Because the economy of pulping processes depends almost entirely on the
efficiency of removing lignin which encrusts the wood fibers. Thus, one way to
improve the efficiency of pulping is to genetically reduce the quantity or to
alter the quality of lignin in pulpwood species. Lignin biosynthesis was the
most peculiar biological process involving various components of wood. Genetic
manipulation of lignin is largely a matter of manipulating genes encoding lignin
pathway-specific enzymes. It takes a long time to reach the breeding goals in
traditional breeding program, because multi-genes controlled most wood traits.
At present, transgenic method is of great expectations. So, the genetic
engineering of lignin biosynthesis has bright prospects in its application in
tree breeding for pulping and papermaking industries. Research in this field
should be strengthened, to breed new varieties for commercial plantations for
pulp in Yangtze and Yellow River systems.

2.5 Safety control

The research on transgenic safety evaluation is very important
for the extension of transgenic trees in China and in the world, and for setting
up the basis for the evaluation systems and standards. Since 2000, Chinese
researchers have studied the effects of transferring Bt gene of
Populus nigra on un-target insects, microbes in soil, mammals, and human
beings, and progress has been made. But because the safety evaluation is a
long-term and complex process, support for these activities should be
strengthened for keeping the leading position in the world.